The invention relates to methods to produce glycan derivatives and particularly to hydroxylamination of glycans.
As interest in the biological role of complex sugars (glycans) develops, it has become increasingly important to have improved methods for the separation and analysis of glycans. A primary problem in the analysis of glycans is the difficulty in detection of the small amounts which are typically available (at the picomol level or less). To this end, methods have become available which employ either radiochemical labelling of the glycans, or the attachment of UV-absorbing or fluorescent labels.
Radiochemical labels are usually introduced by reduction with tritium-labelled borohydride. This approach suffers the disadvantages associated with the use of radioactive materials, but is rigorously quantitative in the sense that, when the reduction is carried to completion, there is a direct correspondence between the molar amounts of different glycan species present in a mixture and the relative amounts of tritium label incorporated.
To date, the standard method of introducing labels has been by reductive amination (RA. FIG. 1), whereby the glycan is converted by an excess of a primary aromatic amine into an acyclic imine, which is stabilised by reduction with cyanoborohydride. This is achieved by heating in an acidic medium, often in the presence of an organic co-solvent, such as dimethyl sulfoxide. The presence of the acid as a catalyst for the derivatisation has a potentially serious disadvantage that it can effect the hydrolysis of labile linkages in the glycans, particularly those of sialic (neuraminic) acids. Moreover, the quantitative aspects of reductive amination of glycans are rather problematic [Suzuki et al. 1992; Evangelista et al. 1996], with considerable potential for the distortion of the quantitative results.
The persistence of problems in the RA of glycans underlines the lack of fundamental understanding of the steps in the overall process, and the lack of appreciation of why it is so slow, where the rate-limiting steps lie and what competing reactions limit the quantitative outcomes. The overall reaction scheme is shown in FIG. 1. In principle, the rate of the overall process could be limited by:
i. the rate of opening of the terminal ring of the reducing glycan (equilibrium 1);
ii. the rate of condensation of the primary amine to form an imine (equilibrium 2);
iii. the rate and equilibrium distribution in the isomerisation to ring forms of the imine (equilibrium 3); or
iv. the rate of reduction of the acyclic imine (step 4).
Each of these steps is subject to acid catalysis, and the improvement in rate in the presence of acid [Evangelista et al. 1996] is not in itself surprising. The degree of acidity required for effective reductive amination of sugars is, however, much greater than that required for simple aldehydes (which have steps 2 and 4 in common with the reaction of the sugar), suggesting that the acid catalysis is required for step 1 or step 3.
Glycan nitrones have been prepared from O-protected D-arabinose derivatives (Paulsen and Budzis, 1974), but not from non-protected monosaccharides and glycans, such as described in this document. The examples of particular relevance have nitrones formed by condensation of the free reducing terminus (the pro-aldehyde at C1) with an N-substituted hydroxylamine. In all such cases, the nitrone product was determined to exist in an open-chained (acyclic) form, on the basis of proton nuclear magnetic resonance (nmr) properties. In this earlier study (Paulsen and Budzis, 1974), the reported nitrones were not submitted to reduction.
For the effective analysis of glycans, including those with acid-labile linkages, it is desirable to have a method which can replace that of reductive amination of primary aromatic amines, and which does not require the inclusion of large amounts of acid to the reaction mixture. The present inventors have developed a new method for producing derivatives of glycans which employs reaction of reducing glycans with mono-N-substituted hydroxylamines, which condense with the glycans to form glycan nitrones which are readily reduced to the stable products. N,N-disubstituted hydroxylamines. The advantage of this method is that the overall reaction can be performed at pH 6 or above, and is so rapid that aldoses can be completely derivatised without necessarily heating for long periods.
In a first aspect, the present invention consists in a method of producing a glycan nitrone comprising reacting the glycan with a mono-N-substituted hydroxylamine at pH 6 or greater to form a glycan nitrone.
In a preferred embodiment of the first aspect of the present invention, the reaction is carried out at pH 6.5 or above.
Mono-N-substituted hydroxylamines suitable for use in the first aspect of the present invention include benzylic N-substituted hydroxylamines and N-arylhydroxylamines.
Benzylic N-substituted hydroxylamines can readily be prepared by reduction of oximes obtained by reaction of aromatic aldehydes with hydroxylamine. In particular, N-(3-methoxylbenzyl)hydroxylamine and N-(3,4-methylenedioxybenzyl)hydroxylamine can be prepared from 3-methoxybenzaldehyde and piperonal, respectively. Benzylic N-substituted hydroxylamines can also be prepared by alkylation of hydroxylamine using an excess of a benzyl halide, with formation of an N,N-disubstituted hydroxylamine, from which one of the aralkyl substituents is removed by mild oxidation followed by hydrolysis.
N-arylhydroxylamines are most conveniently prepared by reduction of corresponding nitro compounds or oxidation of the corresponding primary amines. The success of this approach has been demonstrated by reduction of nitrobenzene to N-phenylhydroxylamine. Alternatively, N-arylhydroxylamines can be prepared by oxidation of the corresponding amines, for instance by using samarium diiodide as oxidant.
In a second aspect, the present invention consists in a method of producing a glycan derivative comprising reacting a glycan nitrone with a reducing agent to form a hydroxylamine glycan derivative.
Preferably, the glycan nitrone is produced by the method according to the first aspect of the present invention.
A reducing agent suitable for this reaction is sodium cyanoborohydride. It will be appreciated, however, that other reducing agents would also be suitable for use in this method.
The method of the present invention allows the inclusion of detectable labels on glycans that are useful in a number of separation and analytical procedures. Furthermore, the glycan derivatives produced by the methods of the present invention can have active moieties introduced therein that are useful for attaching glycans to supports.
The glycan derivatives of the present invention, whether in the form of nitrone or hydroxylamine derivatives, are particularly useful for the analysis or solid-phase immobilisation of glycans and sugars.
The methods of the present invention are also useful for the derivatisation of any carbonyl compound obtained from a glycan or glycoconjugate by any chemical treatment, such as by periodate oxidation or deamination using nitrous acid, or enzymic treatment, such as by treatment with galactose oxidase. Furthermore, the methods of the present invention can be used for the derivatisation of any carbonyl compound obtained from any compound of biological or environmental interest for the purposes of enhancing its separation, purification or detection properties.
The methods of the present invention are particularly useful as a means of attaching to a monosaccharide or glycan a label which confers ultraviolet-absorbing or fluorescent properties which enhance its detection properties, or any other groups which enhance the detection of the monosaccharide or glycan by application of enzymic, chemical or physical reagent or method.
The methods of the present invention are useful as a means of attaching to a monosaccharide or glycan a label which confers modified or improved properties for separation by any method of chromatography, electrophoresis, attachment to a solid support, phase extraction or any other physical, chemical or biological method.
The methods of the present invention are useful as a means of attaching to a monosaccharide or glycan a label which confers modified or improved properties for analysis by any form of mass spectrometry.
A glycan, or a glycoconjugate in which one or more carbonyl groups have been introduced by chemical or enzymic treatment can also be derivatised according to the methods of the present invention.
The methods of the present invention can be used for derivatisation of a reducing glycan, or any modified glycan into which one or more carbonyl groups have been introduced by chemical or enzymic treatment, using reduction at a pH of 6 or above.
A glycan which has been immobilised on a solid support, either as a nitrone, or as a hydroxylamine derivative obtained by reduction of a nitrone, can be used for probing by antibodies, lectins or other reagents, or for the purpose of degradation or any other form of chemical or enzymic modification. Such glycan derivatives can also be used for the purposes of carrying out any form of affinity fractionation or purification of a protein, glycoprotein, glycan or any other biological or biologically-active substance.
The methods of the present invention can be used to label a cell-surface glycan or glycoconjugate, or a cell-surface glycan or glycoconjugate which has been modified either chemically, such as by oxidation by periodate, or enzymically, such as by galactose oxidase, for the purpose of identifying, separating or analysing glycan or glycoconjugate components which are located at the surface of a cell or cells.
The methods of the present invention can be used to label any other cell-surface component, including such components which have been modified by any enzymic or chemical method in order to incorporate an aldehyde or ketone group, for the purpose of identifying, separating or analysing those components which are located at the surface of a cell or cells.
As used herein, the term xe2x80x98glycanxe2x80x99 refers to a monosaccharide, oligosaccharide or polysaccharide containing one or more different monosaccharide subunits. Unless stated otherwise, xe2x80x98glycanxe2x80x99 refers to an unprotected glycan.